This application addresses broad Challenge Area (11): Regenerative Medicine, and specific Challenge Topic 11-AR-101 Musculoskeletal and Skin Regeneration Every year, over a million Americans are hospitalized for bone fractures at a cumulative cost of over $100 billion when considerations such as lost working days are factored in. Large segmental defects are the most clinically challenging types of fracture to manage. Those occurring in the distal tibia, for instance, frequently require multiple procedures to achieve union. Even when successful, the recovery time is considerable and, even under the best of circumstances, the patient often cannot return to full activity for a year or longer. Such injuries are increasingly common because of increased survivability of high energy trauma in civilian settings as well as the continuing military conflicts in Iraq and Afghanistan. Increasingly, large segmental defects are also seen in patients who have required multiple revisions of failed total joint replacements. Current approaches to treating such injuries include the use of autograft and allograft bone, distraction osteogenesis, and the application of recombinant, human bone morphogenetic proteins (BMPs). Each of these has considerable drawbacks. The research described in this proposal aims to develop a novel strategy for healing long bone defects that is more effective and far less expensive than existing methods, and can be accomplished in a single operative procedure. It utilizes a novel device known as the Reamer-Irrigator-Aspirator (RIA) that permits the rapid, straightforward and relatively non-invasive harvest of autologous bone and osteoprogenitor cells from the intramedullary canals of long bones. Our previous research has shown that the progenitor cells recovered by the RIA are more abundant and more osteogenic than marrow cells recovered by traditional bone marrow aspiration. They are highly responsive to rhBMP-2 and we hypothesize that combining the cells and osseous particles recovered by the RIA with rhBMP-2 will generate a powerful, synergistic, osteogenic response. Moreover, the amounts of rhBMP-2 needed to provoke this response are likely to be far lower than those presently used clinically. Because rhBMP-2 is so expensive, this will enormously improve cost-effectiveness. The experiments described in this proposal will evaluate these hypotheses in an athymic rat model. Critical size (5mm), segmental defects will be surgically created in the femora of athymic (nude) rats. These defects do not heal spontaneously. Because the animals are athymic, they will accept human xenografts. Combinations of human osseous particles and marrow cells recovered by the RIA, and rhBMP-2 will be placed into the defects. In Specific Aim 1, we will confirm and extend our preliminary findings of a very marked synergy between these components when implanted into the defects. In Specific Aim 2, the dose of rhBMP-2 will be optimized and time-course experiments carried out to determine the kinetics of bone healing by the optimized combination of BMP-2 and material recovered by the RIA. In these two Specific Aims, healing will be monitored by weekly X-ray until euthanasia at week 8. Post-mortem, femora will be analyzed by dual energy X- ray absorptiometry, 1/4-computed tomography, histology and mechanical testing. Specific Aim 3 will address the contributions of the implanted human cells to healing of the defect. To this end, human marrow cells recovered by the RIA will be stained with a commercial stain, Cell Tracker Orange, just prior to implantation. Sections of the healed rat femora will be stained immunohistochemically with antibodies that recognize human, but not rat, nuclear antigen and thus stain all human nuclei. The identities of any human cells within the healed bone will be further probed using antibodies against human Runx2, to identify human osteoblasts, and against human tartrate-resistant acid phosphatase, to identify human osteoclasts. Because of our prior experience with the core technologies to be used in this project, the proposal is "shovel ready" and we have developed an aggressive, but feasible, timetable. It is based upon the quarterly reporting requirements and will permit the work to be accomplished in 2 years, with clearly identified milestones. Its successful completion will generate considerable additional, sustained activity in the form of human clinical trials, as the technology moves into clinical application, and further pre-clinical research, as the technology is applied to additional problems in the healing of bone and other connective tissues, such as cartilage, meniscus, ligament and tendon. Our project investigates a new way to heal broken bones that should be quicker, more effective and less expensive than existing methods. Before trying this on people, we will undertake a study in rats and we will see whether healing has occurred by looking at X-rays, by determining how strong the bones are, and by examining the bones in other ways. If this project is successful, it will prevent much human suffering, enable people to return to work more quickly, and save money.